ISSN: 1314-6246 Kulac et al. J. BioSci. Biotech. 2018, 7(2): 135-142.

RESEARCH ARTICLE

Semsettin Kulac 1 Physiological, nutritional, and biochemical

Yakup Cikili 2
responses under nickel toxicity in black poplar
Halil Samet 3
Ertugrul Filiz 4 (Populus nigra)
Authors’ addresses: ABSTRACT
1 Duzce University, Faculty of Forestry,
Nickel (Ni) is an essential nutrient for plants and it has been identified as a
Department of Forest Engineering,
Duzce, Turkey. component of a number of enzymes such as ureases. In this study, we have studied
2 Canakkale Onsekiz Mart University,
the long-term effects of nickel toxicity on black poplar (Populus nigra). The black
Faculty of Agriculture, Department of Soil poplars were exposed to Ni as NiSO4.6H2O (200, 400, or 800 µM) for 28 days by
Science and Plant Nutrition, Canakkale,
Turkey. using complete randomized design with three replications. In this context, Ni
3 Kocaeli University, Vocational School accumulation and biomass, photosynthetic pigments analyses [chlorophyll a and b
of Food and Agriculture, Department of (Chl a and b), carotenoid (Car)], malondialdehyde (MDA) content, antioxidant
Crop and Animal Production, Kocaeli, enzyme activities [catalase (CAT) and ascorbate peroxidase (APX)], and metallic ion
Turkey.
4 Duzce University, Cilimli Vocational accumulations were investigated. Ni concentrations significantly increased in root,
School, Department of Crop and bark, and leaves in all Ni treatments. Also, reductions were determined significantly
Animal Production, Duzce, Turkey. in the photosynthetic pigments (Chl a, Chl b, Chl a+b, and Car) at all Ni treatments.
The MDA content, CAT and APX activities significantly increased compared the
Correspondence:
control plants. According to element analyses, the concentration of metallic ion
Ertugrul Filiz
Duzce University, Cilimli Vocational accumulations [potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), iron
School, Department of Crop and Animal (Fe), zinc (Zn), manganese (Mn), and copper (Cu)] were affected by Ni exposures,
Production, Duzce, Turkey. suggesting that Ni toxicity adversely affects physiological activities in P. nigra.
Tel: +90 5058735820
Fax: +90 3806817313
e-mail: [email protected] Key words: phytotoxicity, metallic ion accumulation, heavy metal, plant stress

Article info:
Received: 2018
Accepted: 2018

2009). The absorption and translocation of Ni2+ from roots to

Introduction shoots regulated by the inhibitory effect of various metal ions
Nickel (Ni) that are found in soil, water and air samples varied as Fe3+>Co2+>Ca2+>Mg2+>NH4+>K+>Na+ (Temp
within the biosphere is one of the trace metals emitted into 1991). Ni is one of the heavy metals and essential
the environment by anthropogenic and natural sources microelements for plant metabolism and a major component
(WHO, 1991). Ni is found abundantly as a free metal or a of plant enzymes such as urease (EC 3.5.1.5, urea
complex with iron (Fe) in igneous rocks. Also, Ni is the 22 nd amidohydrolase), Ni-dependent metalloenzyme (Sreekanth et
most abundant elements in the earth's crust (Sunderman and al., 2013; Filiz et al., 2016). While Zn2+, Cu2+, Co2+, Cd2+,
Oskarsson, 1991). Ni is mostly present in the form of and Pb2+ inhibited Ni2+ influx in barley roots, Mn2+ did not.
nickelous ion, Ni2+ in nature and hydrated form as Ni In addition, Zn2+ and Cu2+ were strongly competitive with
(H2O)62+ is common form observed in the soil solution Ni2+ (Körner et al., 1987). As with many heavy metals, Ni
(Yusuf et al., 2011). The uptake of Ni in plants is performed disturbs various physiological processes in plants, containing
by the root system with passive diffusion and active transport enzyme activities (Van Assche and Glijsters, 1990). Ni plays
(Seregin and Kozhevnikova, 2006). The general uptake of Ni important roles in growth and development of plants such as
by plants is affected by plant metabolism, the presence of seed germination, seedling, root, leaf, and stem growth, and
other metals and organic matter composition, the acidity of total dry matter production. Also, the toxic effects of Ni were
soil or solution, and the concentration of Ni2+ (Chen et al., detected in various physiological processes, including

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photosynthesis, water relations, mineral nutrition, and effect Determination of Ni and ion contents
on metabolites. The enzymes and other cellular processes After four weeks of Ni treatment, the shoots and roots
were affected by Ni toxicity such as nitrate reductase (NR), were carefully harvested, washed with running tap water, and
nitrogen metabolism, plasma membrane H+-ATPase, then rinsed three times with de-ionized water. The all shoots,
glutathione reductase (GR), and oxidative stress and barks, and roots were oven dried at 70°C for at least three
antioxidant systems (Yusuf et al., 2011). In white birch days, and the dry weight (DW) was immediately measured.
(Betula papyrifera), Ni and copper (Cu) toxicity was The shoots samples were ground to powder for nutrient ions
evaluated by using physiological and genomics approaches analysis. Then, these samples were digested by using dry ash
(Theriault and Nkongolo, 2016). Fuentes et al. (2007) method for extractions in a muffle furnace at 500°C for 6
reported that heavy metal concentration increased and was hours (Miller 1998). The Ni and nutrient ions were
always higher in roots than in shoots under Ni, Cu, and Zn determined by ICP-OES (Perkin-Elmer Optima 2100 DV;
exposure in Pinus halepensis, Pistacia lentiscus, Juniperus Waltham, MA). The bio-concentration factor (BCF),
oxycedrus, and Rhamnus alaternus. In P. nigra, leaf Ni translocation factor (TF), and total accumulation rate (TAR)
content was found as lower in mature than in developing were calculated according to the following formulas (Ait Ali
leaves and Ni stress significantly decreased photosynthesis et al., 2002; Shi et al 2010; Çikili et al., 2016):
(Velikova et al., 2011). In this study, it was aimed to BCF = [Cd ]shoot or root / [total Cd]growth medium
investigate the physiological and biochemical responses of P.
TF (%) = 100 x [Cd]shoot / [Cd]root
nigra under excess Ni.
TAR of Cd (μg/g DW /day) = ([Cd]shoot x DWshoot) +
([Cd]root x DWroot) / growth day x (DWshoot + DWroot)
Materials and Methods
Determination of photosynthetic pigments
Cultivation of plants and Ni treatment
The chlorophyll a (Chl a), chlorophyll b (Chl b), and
The experiments were performed in the greenhouse under
carotenoids (Car) concentration were determined
natural light conditions at an ambient temperature 70%
spectrophotometrically (Shimadzu UV-1201; Tokyo) by
average relative humidity and 25/18°C day/night average
using 500 mg fresh weight (FW) of leaf material ground in a
temperature in Cilimli Vocational School of Duzce
homogenizer in the presence of 10 mL of acetone 90% (v/v).
University, Turkey (lat. 40°53ʹ40ʹʹN, long. 31°02ʹ55ʹʹE), in
The absorbance of the extract was measured at 663, 645, and
2016 June. Cuttings (nearly 15 cm in length and 1 cm in
470 nm and pigment concentrations were calculated
diameter) of black poplar (P. nigra genotype Gazi) were
according to Lichtenthaler (1987).
taken from Poplar and Fast Growing Forest Trees Research
Institute, Izmit, Turkey. One cutting with a sprout was Malondialdehyde content and membrane permeability
planted and rooted in pots for 10 weeks. Later, these cuttings The level of lipid peroxidation in leaves was determined
were transplanted at a rate of one plant per pot filled with 3 according to Hodges et al., (1999) by measuring
liters of perlite. The poplar cuttings were grown in two weeks malondialdehyde (MDA), routinely used as an indicator of
by using quarter-strength, one week by half-strength and one membrane lipid peroxidation, using 250 mg of fresh tissue
week full-strength Hoagland solution for each day. The homogenize with 5% (w/v) trichloroacetic acid (TCA). Lipid
modified Hoagland solution consisted of 5 mM KNO3, 5 mM peroxidation products were measured as the content of
Ca(NO3).4H2O, 2 mM MgSO4.7H2O, 1 mM KH2PO4, 45.5 thiobarbituric acid (TBA)-reactive substances. Membrane
µM H3BO3, 44.7 µM FeSO4.7H2O, 30.0 µM NaCl, 9.1 µM permeability (MP) measurements using fresh matter were
MnSO4.H2O, 0.77 µM ZnSO4.7H2O, 0.32 µM CuSO4.5H2O, done before harvest. Membrane permeability was determined
0.17 µM NiSO4.6H2O, 0.10 µM (NH4)2Mo7O24.4H2O and for the shoot disc samples by the electrical conductivity
54.8 µM Na2EDTA.2H2O adjusted to pH 6.0 After an (EC%) method (Yan et al., 1996).
acclimatization period of 4 weeks, cuttings with a similar
Antioxidant enzyme extraction and assay
number of nodes and height were chosen for the experiment.
For Ni treatments, four levels of Ni (0, 200, 400 and 800 µM) For extraction and assay of enzymes, fully matured leaves
as NiSO4.6H2O were applied to the perlite. The experiment (1.0 g) were homogenized (Heidolph, Diax 900) with 5 mL
was designed as a complete randomized design with three of extraction buffer (100 mM Na-phosphate buffer, pH 7.5)
replications. Ni treatment to the plants continued for 28 days. containing 0.5 mM EDTA-Na2 at 4ºC. Also, 1 mM ascorbate
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was included to extraction buffer for ascorbate peroxidase treatments significantly increased Ni contents in all parts of
due to the instability of APX in the absence of ascorbate. The plant due to dose levels (Table 1). While the Ni concentration
homogenate was centrifuged at 10 000 g for 5 min. The at 800 µM Ni as the highest level treatment was about 940,
supernatant was used for determining the enzymes activity 272, and 578 folds higher in leaves, bark, and roots,
analyses and a spectrophotometer (Shimadzu UV/VIS 1201, respectively compared to control plants, it was seen about
Japan) was used for all colorimetric measurements (including 304, 70, and 216 folds higher in leaves, bark, and roots,
enzyme activities) at 25ºC. The activity of CAT (EC 1. respectively at 200 µM Ni as the lowest level treatment.
11.1.6) was determined by using a reaction solution Velikova et al. (2011) reported that Ni contents dramatic
containing 50 mM potassium dihydrogen phosphate increased 30 and 200 µM treatments in P. nigra. In
(KH2PO4) and 1.5 mM H2O2 as a decrease in absorbance at Mediterranean woody seedlings (P. halepensis, P. lentiscus,
240 nm for 1 min following the decomposition of H 2O2 and J. oxycedrus, and R. alaternus), Ni concentration
calculated using the extinction coefficient (E = 40 mM/cm) significantly increased under 25 and 50 µM of Ni exposures
for H2O2 (Cakmak et al., 1993). The activity of APX (EC (Fuentes et al., 2007). These data are in agreement with our
1.11.1.11) was determined by using a reaction solution results. Also, Ni treatments inhibited the root, leaf, and bark
containing 50 mM KH2PO4, 0.05 mM ascorbic acid, 0.1 mM dry weights in black poplar for all Ni levels but not
EDTA-Na2, and 1.5 mM H2O2 as a decrease of ascorbate and significant (Table 1). Fuentes et al. (2007) indicated that
measuring the change in absorbance at 290 nm for 1 min and intermediate application rates of Ni showed a positive trend
calculated using the extinction coefficient (E = 2.8 mM/cm) on biomass accumulation in Mediterranean woody seedlings
for ascorbate (Nakano and Asada, 1981). (P. halepensis, P. lentiscus, J. oxycedrus, and R. alaternus) at
25 and 50 µM. The total dry matter production and yield
Statistical analyses
were significantly affected by nickel and Ni can also disturb
The experimental design was a completely randomized some plant physiological processes, such as mineral nutrition,
factorial design with three replicates and obtained data were photosynthesis, and water relations (Sreekanth et al., 2013).
analyzed by ANOVA. The differences were compared by the In this study, the decrease of dry weights may be related to
Tukey HSD test (P≤0.05) and performed by using the JMP these adverse effects of Ni treatments at high doses on plant
package program (SAS Institute Inc., Cary, NC). metabolism.

Results and Discussion Ni effects on photosynthetic pigments

The photosynthetic pigments content was significantly
Ni accumulation and biomass
decreased in leaves of treated plants (Table 2). The content of
Ni accumulation in black poplar was determined four Chl a, Chl b, Chl a+b and Car decreased dramatically in
weeks after Ni exposure. The results indicated that Ni leaves at all levels of Ni treatments. At the highest dose as

Table 1. Total Ni concentration and dried weights of roots, leaves, and bark in P. nigra exposed to NiSO4.6H2O for four
weeks.
Leaf Ni Bark Ni Root Ni
Ni treatments Leaf DW Bark DW Root DW
concentration concentration concentration
(µM) (g/plant) (g/plant) (g/plant)
(μg/g DW) (μg/g DW) (μg/g DW)
0 0.2±0.04 d 0.7±0.12 d 4.8±0.06 c 5.59±0.38 1.79±0.26 1.90±0.27
200 67.1±6.15 c 54.4±2.03 c 1032.8±13.5 b 4.69±0.41 1.66±0.13 1.57±0.22
400 158.6±0.55 b 125.4±3.47 b 2320.1±268 a 5.25±0.22 1.76±0.18 1.83±0.01
800 188.2±1.20 a 193.3±6.50 a 2758.5±183 a 4.54±0.29 1.39±0.15 1.44±0.04

Tukey HSD0.05 14.2 17.3 735.9 ns ns ns

Data are means of three replicates with standard errors (means ± SE, n=3). Different letters on the bars indicate statistically
significant differences (P≤0.05) between the treatments according to Tukey’s HSD test. ns: not significant
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Table 2. The content of photosynthetic pigments under Ni exposure at different concentrations.

Ni treatments Chl a Chl b Chl a+b Car
Chl a/b Car/Chl
(µM) (µg/g FW)
0 1069±53 a 267±6 a 1336±49 a 665±13 a 4.01±0.28 0.499±0.01 b
200 963±26 a 249±13 a 1212±38 a 625±21 a 3.88±0.10 0.516±0.01 b
400 484±59 b 112±13 b 595±71 b 321±36 b 4.33±0.17 0.541±0.01 ab
800 358±8 b 102±15 b 460±14 b 268±31 b 3.66±0.50 0.583±0.02 a

Tukey HSD0.05 190 60 220 70 ns 0.06

Data are means of three replicates with standard errors (means ± SE, n=3). Different letters on the bars indicate statistically
significant differences (P≤0.05) between the treatments according to Tukey’s HSD test. ns: not significant

The bio-concentration and translocation factors, and total

800 µM Ni treatment, Chl a, Chl b and Car declined by accumulation rate
nearly 66%, 63%, 65% and 59%, respectively. Nevertheless, BCFs, TFs, and TAR values were determined and shown
Car/Chl was remarkably increased with Ni treatments. In four in Table 3. The BCF is used for evaluating the metal
white poplar (P. alba) clones (Villafranca, L-12, L-80, and accumulation efficiency in plants. The BCF values in leaves,
LBM), higher concentrations of Ni in the growth medium bark, and root remarkably decreased in all Ni treatments
showed significant inhibitory effects on plant fresh mass and compare to control plant. The present results showed that
especially on the photosynthetic pigments content (Katanić et BCF value of black poplar in leaves, bark, and root was
al., 2015). Heavy metals such as nickel affect the structure greater than critical level (BCF>1) for hyper-accumulator
and function of the chlorophyll molecule by displacing the plants as accepted by Ma et al. (2001), and therefore black
Mg atom in the center of chlorophyll molecule (Van Assche poplar could be a Ni hyper-accumulator plant. Hyper-
and Clijsters, 1986; Kupper et al., 1996). Seregin and Ivanov accumulators have a greater absorbance capacity (50-500-
(2001) reported that heavy metals disrupted chloroplast fold more) than normal plant, because of having root-to-shoot
structure, blocked chlorophyll synthesis, and disordered transport system and increased detoxification capacity
electron transport. Thus, our results are in agreement with (McGrath and Zhao, 2003).
these data.
The TF is a crucial term to understand the ability of plants
to translocate heavy metals from roots to shoot (Zacchini et

Table 3. Effects of Ni treatments on bio-concentration and translocation factors and total accumulation rate of nickel in P.
nigra.
Ni treatments Leaf Bark Root Leaf TF Bark TF TAR
(µM) BCF BCF BCF (%) (%) (μg/g DW/ day)
0 21.93±0.00 a 70.70±0.00 a 476.9±0.0 a 4.59±0.82 14.87±2.70 a 3.9±0.7 d
200 5.71±0.52 b 4.63±0.17 b 88.0±1.2 bc 6.49±0.60 5.26±0.22 b 579.5±85.8 c
400 6.75±0.02 b 5.34±0.15 b 98.8±11.1 b 7.02±0.82 5.58±0.78 b 1660.0±93.5 a
800 4.01±0.03 b 4.12±0.14 b 58.7±3.9 c 6.88±0.44 7.05±0.33 b 1335.6±49.1 b

Tukey HSD0.05 9.10 27.35 32.26 ns 6.40 308

Data are means of three replicates with standard errors (means ± SE, n=3). Different letters on the bars indicate statistically
significant differences (P≤0.05) between the treatments according to Tukey’s HSD test. ns: not significant.
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al., 2011). In leaves, TF values increased but not significant, environmental stresses cause to excessive production of ROS
whereas TF values significantly decreased by about 65%, resulting oxidative damage and ultimately cell death.
63%, and 53% at 200, 400, and 800 µM Ni treatments, Scavenging or detoxification of excess ROS are realized by
respectively in bark tissue. The TF in leaves and bark was an efficient antioxidative enzyme system, containing
much lower than the critical level (TF> 100%). Also, TAR superoxide dismutase (SOD), catalase (CAT), guaiacol
values significantly increased depending on Ni treatment peroxidase (GPX), ascorbate peroxidase (APX) etc. (Noctor
dose. While the highest TAR value was found at 400 µM Ni and Foyer, 1998; Sharma et al., 2012). Hao et al., (2006)
treatment (about 426 folds) as compared to control, the showed that excessive Ni causes significant increases in the
lowest value was identified at 200 µM Ni treatment (about concentration of hydroxyl radicals, superoxide anions, nitric
149 folds). oxide and hydrogen peroxide. In this study, the CAT and
APX activities enhanced significantly in leaves under Ni
MDA and membrane permeability
treatments (Figure 2). While the CAT activity increased by
MDA is widely used as an index for the status of lipid about 21%, 34%, and 55% at 200, 400, and 800 µM
peroxidation. In this study, lipid peroxidation level in leaves treatments, respectively, the APX activity enhanced by about
was measured as MDA content, was given in Figure 1. At the 17%, 34%, and 36% at 200, 400, and 800 µM Ni treatments,
highest Ni treatment (800 µM Ni), the highest increase in respectively. Thus, CAT activity was found as higher than
MDA content was identified as 18% compared to control APX activity in black poplar. It can be suggested that CAT
plants. In 200 µM and 400 µM Ni treatments, increases of activity may plays a more active role than APX to combat
MDA content were found as about 6% and 7%, respectively, oxidative stress because under Ni stress in P. nigra. It has
indicating that Ni toxicity induces oxidative stress. In been previously reported that Ni can increase the activities of
membrane permeability, increases were detected in leaves for SOD, POD and APX (Gajewska and Skłodowska, 2008).
all levels of Ni treatments but not significant (Figure 1). The Seregin and Kozhevnikova (2006) reported that most of
Ni2+ affected the sterol and phospholipid composition of the enzyme activities were decreased, in contrast to some
plasma membrane in rice and adversely affects the membrane activities such as antioxidant enzymes increased at high Ni
permeability and ion homeostasis in the cytoplasm (Ros et concentrations. These data are in agreement with our
al., 1990). In reviews of Ni metabolism in plants, Ni stress findings.
enhanced MDA concentration, disturbed membrane
functionality, water balance, and ion balance in the cytoplasm
in different plant species (Seregin and Kozhevnikova, 2006;
Yusuf et al., 2011; Sreekanth et al., 2013). In the current
study, these symptoms were detected in black poplar under
Ni stress conditions.

Figure 2. Effects of different Ni concentrations on CAT

and APX activity in leaves of P. nigra. Results are means
± SE of three independent replicates. Different letters are
significant at P≤0.05 according to Tukey’s HSD test.

Metallic Cation Accumulations

Figure 1. Effects of different Ni concentrations on MDA The heavy metal toxicity can affect the many cellular
content and MP in leaves. Bars indicate means of three processes and one of them is the reduction of cation and
replicates ± SE. Different letters on the bars indicate anion absorption by plant roots (Pallavi and Ram Shankar,
significant difference (P≤0.05) between the treatments 2005). The Ca, Mg, Mn, Fe, Cu, and Zn show the similar
according to Tukey’s HSD test.
characteristics to Ni, thus Ni could compete with these
Effect of Ni on antioxidative enzyme activities minerals in absorption, uptake and utilization in the plant
metabolism (Chen et al., 2009). Therefore, this could lead to
Reactive oxygen species (ROS) are produced in plant
perturbation in some physiological and biochemical
cellular metabolism as a normal product. The different
processes, and finally concludes in phytotoxic damages
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(Gajewska et al., 2006). In this study, metallic ion (2012) reported that the nutrient reserves followed the
accumulations such as K, Ca, Mg, Na, Fe, Zn, Mn, and Cu sequence Ca>K>Mg>Si>Na in bark in poplar clones Robusta
were investigated under excessive Ni conditions in leaves, (Populus x euramericana) and I-214 (Populus x
bark, and roots of P. nigra (Table 4 and 5). In roots, euramericana). In this study, the nutrient reserves were found
accumulations of K, Ca, Na, Fe, Zn, and Cu significantly as K>Ca>Mg>Na in the bark, suggesting that genetic
decreased compared to control plants, except for Cu background and environmental conditions may affect the
concentration at the highest Ni treatment, whereas Mn differences in metal ion concentrations in P. nigra. The
accumulation enhanced in all levels of Ni treatments. In reduction in uptake of Mg and Fe is one of the major causes
leaves, K, Mg, and Na accumulations significantly increased, of chlorosis induced by excess of environmental Ni (Piccini
in contrast, declines of Zn accumulation was determined. and Malavolta 1992). At high Ni concentrations (about 0.1–1
Also, an increment in Ca and Fe accumulations and decreases mM), the contents of macro and micro-nutrients are
in Mn and Cu accumulations were observed but not negatively affected because of the perturbations in absorption
significant. In bark, Mg, Fe, Zn, Mn, and Cu accumulations and transport (Rubio et al., 1994). In barley, reductions in Ca,
significantly decreased whereas Na accumulation Fe, K, Mg, Mn, P, and Zn contents were determined under
significantly enhanced at 800 µM Ni treatment. Petráš et al. toxic concentration of Ni in leaves and roots (Brune and

Table 4. The changes in K, Ca, Mg, and Na concentrations under Ni exposures in P. nigra.
Ni treatments K Ca Mg Na
(µM) (mg/g DW) (mg/g DW) (mg/g DW) (mg/g DW)
Leaves
±0.7 ±0.06
0 24.8 b 4.55 a 3.88±0.07 b 0.31±0.03 b
200 28.7±0.2 a 4.86±0.07 a 4.37±0.02 a 0.33±0.02 b
400 25.5±0.2 b 5.16±0.21 a 4.58±0.08 a 0.43±0.04 ab
800 25.9±0.4 b 5.14±0.13 a 4.34±0.05 a 0.53±0.06 a
Tukey HSD0.05 1.87 0.61 0.25 0.16
Bark
±0.6 ±0.06
0 32.3 20.77 ab 8.82±0.08 a 0.90±0.10 b
200 35.7±1.2 21.61±0.07 a 8.14±0.14 b 0.88±0.01 b
400 34.8±0.4 20.67±0.21 ab 8.08±0.07 bc 0.96±0.01 ab
800 34.9±1.0 18.57±0.13 b 7.66±0.04 c 1.21±0.06 a
Tukey HSD0.05 ns 2.59 0.42 0.29
Root
0 26.8±0.4 a 10.91±0.17 a 6.81±0.05 ab 10.32±0.14 b
200 25.5±0.3 a 8.49±0.23 b 6.17±0.12 c 11.06±0.33 ab
400 21.6±0.5 b 7.94±0.22 b 7.14±0.02 a 11.66±0.04 a
800 17.7±0.3 c 8.12±0.01 b 6.55±0.16 bc 7.42±0.21 c
Tukey HSD0.05 1.70 0.80 0.48 0.96
Data are means of three replicates with standard errors (means ± SE, n=3). Different letters on the bars indicate statistically
significant differences (P≤0.05) between the treatments according to Tukey’s HSD test. ns: not significant

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Table 5. The changes in Fe, Zn, Mn, and Cu concentrations under Ni exposures in P. nigra.
Ni treatments Fe Zn Mn Cu
(µM) (µg/g DW) (µg/g DW) (µg/g DW) (µg/g DW)
Leaves
±2.69 ±0.53
0 84.10 25.66 a 30.66±0.43 5.08±0.29
200 91.10±0.63 15.86±0.95 c 28.72±1.36 4.21±0.18
400 91.18±5.32 17.43±0.81 c 28.30±0.92 4.59±0.20
800 92.12±1.17 22.16±0.50 b 28.98±0.63 4.32±0.12
Tukey HSD0.05 ns 3.26 ns ns
Bark
0 74.62±0.87 a 47.68±4.21 a 26.45±0.85 a 16.40±1.89 a
200 69.01±1.18 a 30.81±2.33 b 21.51±1.76 b 10.95±1.37 ab
400 54.48±1.76 b 26.92±0.32 b 15.98±0.46 c 9.22±0.90 b
800 58.85±1.00 b 29.05±0.46 b 15.27±0.16 c 11.22±0.15 ab
Tukey HSD0.05 5.63 10.94 4.54 5.70
Root
±30.1 ±2.20
0 461.32 a 28.09 a 21.03±0.69 b 15.06±0.54 b
200 238.15±15.2 b 21.51±0.45 b 26.55±0.41 a 12.36±0.18 c
400 182.15±5.54 b 17.85±0.44 bc 28.64±0.68 a 12.67±0.47 c
800 164.45±7.72 b 15.88±0.26 c 30.36±2.08 a 17.46±0.33 a
Tukey HSD0.05 79.30 5.22 5.25 1.85
Data are means of three replicates with standard errors (means ± SE, n=3). Different letters on the bars indicate statistically
significant differences (P≤0.05) between the treatments according to Tukey’s HSD test. ns: not significant

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Deitz, 1995). Based on previous data, dramatic reductions
composition of roots and leaves of barley seedlings grown in the
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